Search

Physiological activity of G protein gated inward rectifier K+ (GIRK, Kir3) channel, dynamically regulated by three key ligands, phosphoinositol-4,5-bisphosphate (PIP2), Gβγ, and Na+, underlies cellular electrical response to multiple hormones and neurotransmitters in myocytes and neurons. In a reducing environment, matching that inside cells, purified GIRK2 (Kir3.2) channels demonstrate low basal activity, and expected sensitivity to the above ligands. However, under oxidizing conditions, anomalous behavior emerges, including rapid loss of PIP2 and Na+-dependent activation and a high basal activity in the absence of any agonists, that is now paradoxically inhibited by PIP2. Mutagenesis identifies two cysteine residues (C65 and C190) as being responsible for the loss of PIP2 and Na+-dependent activity and the elevated basal activity, respectively. The results explain anomalous findings from earlier studies and illustrate the potential pathophysiologic consequences of oxidation on GIRK channel function, as well as providing insight to reversed ligand-dependence of Kir and KirBac channels. Graphical Abstract [ABSTRACT FROM AUTHOR]

Muscarinic toxins (MTs) are natural toxins produced by mamba snakes that primarily bind to muscarinic acetylcholine receptors (MAChRs) and modulate their function. Despite their similar primary and tertiary structures, MTs show distinct binding selectivity toward different MAChRs. The molecular details of how MTs distinguish MAChRs are not well understood. Here, we present the crystal structure of M1AChR in complex with MT7, a subtype-selective anti-M1AChR snake venom toxin. The structure reveals the molecular basis of the extreme subtype specificity of MT7 for M1AChR and the mechanism by which it regulates receptor function. Through in vitro engineering of MT7 finger regions that was guided by the structure, we have converted the selectivity from M1AChR toward M2AChR, suggesting that the three-finger fold is a promising scaffold for developing G protein–coupled receptor modulators. [ABSTRACT FROM AUTHOR]

Heterotrimeric G proteins are categorized into four main families based on their function and sequence, Gs, Gi/o, Gq/11, and G12/13. One receptor can couple to more than one G protein subtype, and the coupling efficiency varies depending on the GPCR-G protein pair. However, the precise mechanism underlying different coupling efficiencies is unknown. Here, we study the structural mechanism underlying primary and secondary Gi/o coupling, using the muscarinic acetylcholine receptor type 2 (M2R) as the primary Gi/o-coupling receptor and the β2-adrenergic receptor (β2AR, which primarily couples to Gs) as the secondary Gi/o-coupling receptor. Hydrogen/deuterium exchange mass spectrometry and mutagenesis studies reveal that the engagement of the distal C-terminus of Gαi/o with the receptor differentiates primary and secondary Gi/o couplings. This study suggests that the conserved hydrophobic residue within the intracellular loop 2 of the receptor (residue 34.51) is not critical for primary Gi/o-coupling; however, it might be important for secondary Gi/o-coupling. G protein-coupled receptors (GPCRs) can couple to more than one G protein subtype, and the coupling efficiency varies depending on the GPCR-G protein pair. Here authors use hydrogen/deuterium exchange mass spectrometry and mutagenesis to study the structural mechanism underlying primary and secondary Gi/o coupling. [ABSTRACT FROM AUTHOR]

The article discusses research on the role of muscarinic acetylcholine receptors as G protein-coupled receptors (GPCRs). Topics include the role of iperoxo binding pockets in the receptors M1R and M2R, the role of chimeric receptors and site-directed mutagenesis in research on muscarinic receptors, and the structure of a nucleotide-free G-protein subtype.

The activation of the G-protein transducin (Gt) by rhodopsin (Rho) has been intensively studied for several decades. It is the best understood example of GPCR activation mechanism and serves as a template for other GPCRs. The structure of the Rho/G protein complex, which is transiently formed during the signaling reaction, is of particular interest. It can help understanding the molecular details of how retinal isomerization leads to the G protein activation, as well as shed some light on how GPCR recognizes its cognate G protein. The native Rho/Gt complex isolated from bovine retina suffers from low stability and loss of the retinal ligand. Recently, we reported that constitutively active mutant of rhodopsin E113Q forms a Rho/Gt complex that is stable in detergent solution. Here, we introduce methods for a large scale preparation of the complex formed by the thermo-stabilized and constitutively active rhodopsin mutant N2C/M257Y/D282C(RhoM257Y) and the native Gt purified from bovine retinas. We demonstrate that the light-activated rhodopsin in this complex contains a covalently bound unprotonated retinal and therefore corresponds to the active metarhodopin II state; that the isolated complex is active and dissociates upon addition of GTPγS; and that the stoichiometry corresponds to a 1∶1 molar ratio of rhodopsin to the heterotrimeric G-protein. And finally, we show that the rhodopsin also forms stable complex with Gi. This complex has significantly higher thermostability than RhoM257Y/Gt complex and is resistant to a variety of detergents. Overall, our data suggest that the RhoM257Y/Gi complex is an ideal target for future structural and mechanistic studies of signaling in the visual system. [ABSTRACT FROM AUTHOR]

Highlights: [•] Recent methodologies have rendered structure determination of GPCR more feasible. [•] Interdisciplinary collaboration has promoted the structural biology of GPCR. [•] Growing number of GPCR structures in different states has become available. [•] The structure of an active GPCR and G-protein complex has been determined. [Copyright &y& Elsevier]

Binding of arrestin to phosphorylated G protein-coupled receptors (GPCRs) is crucial for modulating signaling. Once internalized, some GPCRs remain complexed with β-arrestins, while others interact only transiently; this difference affects GPCR signaling and recycling. Cell-based and in vitro biophysical assays reveal the role of membrane phosphoinositides (PIPs) in β-arrestin recruitment and GPCR-β-arrestin complex dynamics. We find that GPCRs broadly stratify into two groups, one that requires PIP binding for β-arrestin recruitment and one that does not. Plasma membrane PIPs potentiate an active conformation of β-arrestin and stabilize GPCR-β-arrestin complexes by promoting a fully engaged state of the complex. As allosteric modulators of GPCR-β-arrestin complex dynamics, membrane PIPs allow for additional conformational diversity beyond that imposed by GPCR phosphorylation alone. For GPCRs that require membrane PIP binding for β-arrestin recruitment, this provides a mechanism for β-arrestin release upon translocation of the GPCR to endosomes, allowing for its rapid recycling. [Display omitted] • β-Arrestins coincidentally detect membrane PIPs for recruitment to GPCRs • GPCR phosphorylation affects dependence on membrane PIPs for β-arrestin recruitment • Membrane PIPs are allosteric modulators of GPCR-β-arrestin complexes • Plasma membrane PIPs alone promote β-arrestin activation By acting as allosteric regulators of arrestin conformation, membrane phosphoinositides potentiate an active conformation of arrestin and stabilize GPCR-arrestin complexes. Membrane PIPs can therefore drive GPCR conformational diversity beyond what is seen from GPCR phosphorylation alone and provide spatial control for GPCR-arrestin complex formation. [ABSTRACT FROM AUTHOR]